Aerobic granules were discovered around 20 years back. These are spherical aggregates of mixed microbial culture, typically around 0.5–3 mm in diameter. Aerobic granule-based wastewater treatment technology has been successfully used on a full-scale for real wastewater treatment. It is argued that the aerobic granule-based technology can potentially replace the traditional activated sludge process. The first of its advantages is that aerobic granule-based wastewater treatment plants can be built within around one quarter of the land occupied by conventional activated sludge-based plants. Additionally, this technology can reduce the operating cost by nearly 25%. Reduction in sludge production and reduction in energy consumption (around 30%) are other benefits of this technology. Granule formation is a relatively slow process. In fact, the time taken for granule formation is a challenge for a full-scale application of this technology. The mechanism behind aerobic granule formation is not fully understood. Thus, further investigation of the molecular mechanism of granule formation would be crucial for the successful commercialization of this technology.

Water impact

The aerobic granulation technology has many advantages over the conventional activated sludge process of wastewater treatment. It can reduce the cost and energy requirement for wastewater treatment. Without reducing the daily wastewater handling capacity, this new technology can reduce the size of the wastewater treatment plant by 75%.

Introduction

Compared to the activated sludge process, aerobic granulation is a relatively new technology for wastewater treatment. Extensive research work on this subject started in the late 1990s.1–4 Aerobic granules are auto-immobilized microspheres of mixed microbial species. They are generated during wastewater treatment using an aerobic sequencing batch reactor. Aerobic granules are denser and heavier than small microbial flocs found in the conventional activated sludge process. These granules can settle down faster than microbial flocs.1,5,6 This helps in the quick separation of the sludge from the treated liquid fraction of the wastewater. The conventional wastewater treatment process uses polymeric flocculants for enhanced sludge settling.7 Owing to their fast settling ability; aerobic granules can be effectively separated from the wastewater without the need for any flocculating agents. It has been reported that aerobic granules may have a specific gravity ranging from 1.004 to 1.1kg dry weight per m3, and this value is significantly higher than around 1.002 to 1.006g dry weight per m3 of activated sludge flocs.8 Likewise, the conventional activated sludge process for wastewater treatment uses large secondary clarifier tanks to hold the wastewater for as long as 12 hours to separate the sludge by gravimetric settling. In contrast, if aerobic granules are used for wastewater treatment, the same reactor can be used for both treatment and clarification. For instance, a SVI30 (sludge volume index) of 80 to 120 is considered appropriate for an activated sludge process and this value is usually lower than its SVI5. However, the SVI30 of aerobic granules could be around 47 and this value is usually similar to its SVI5. This indicates the excellent settling ability of aerobic granules. It would save the land and capital required for constructing the clarifier tanks. Thus, by switching to the aerobic granulation technology, the land requirement for the construction of a new wastewater treatment plant could be reduced by nearly 75%. Reduction in sludge/biomass production is another advantage of aerobic granules.9,10 The cost of sludge dewatering and transportation for final disposal is a major contributor of the total wastewater treatment cost.11 Thus, reduction in sludge production directly reduces the total treatment cost. This new technology can reduce the energy requirement for wastewater treatment by 30% and the overall operation cost is reduced by 25%.12,13

The basic mechanism behind aerobic granule formation is a subject of current fundamental research even after nearly 20 years since its discovery. Numerous hypotheses have been proposed to explain the underlying mechanism of granule formation. However, these mechanisms are not well-established.14 This article discusses the recent theories on granule formation and provides a roadmap for further fundamental investigations to explore its underlying mechanism. Similarly, it discusses the role of reactor design and process parameters on granule formation and wastewater treatment. Aerobic granules are known to have nitrogen and phosphorus removal mechanisms different from that of the conventional activated sludge. Thus, this article discusses recent theories on nitrogen and phosphorus removal mechanisms of aerobic granules. Aerobic granules have been successfully used for biodegradation of organic pollutants and heavy metal removal from wastewater. Recent advances on this subject have also been discussed. There are certain examples of the full-scale implementation of aerobic granules for real wastewater treatment. Such reports have been evaluated to determine the actual potential of this technology. Finally, the challenges of real application of this technology and their potential solutions have been summarized. Thus, it is a comprehensive review of all aspects of aerobic granulation technology for wastewater treatment.

Aerobic granule formation mechanism

As mentioned in the introduction section, there are different hypotheses to explain aerobic granule formation mechanism. However, there is no convincing experimental evidence to support any one of such hypotheses. Recently, we have proposed a new hypothesis of aerobic granule formation. According to this hypothesis, granule formation is initiated by newly germinated fungal spores.15 An initial fungal hyphae matrix could be developed either by a newly germinated fungal spore or by a small fragment of fungal hyphae detached from an already established matrix. Bacteria have gradually started to colonize within a newly developed fungal hyphae matrix. Thus, instead of developing into a proper fungal pellet, the matrix developed into an aerobic granule dominated by bacteria.

Barr et al. (2010) have suggested that there could be two distinct mechanisms behind aerobic granule formation. Compact and smooth granules could be formed by a gradual growth of a single colony of microorganisms. Whereas, relatively loose granules could be developed by aggregation of many independent colonies of microorganisms.16 Usually, the reactor operated under a sequencing batch mode is suitable for aerobic granule formation. Recently, Wu et al. (2015) have investigated the granule formation mechanism under continuous flow conditions. The authors have concluded that a high organic loading rate and a high selection pressure are two crucial factors for aerobic granules formation under continuous flow.17 Another hypothesis suggests that precipitated metal ions such as Fe3+ are responsible for the formation of an inorganic substratum on which microorganisms are attached to form the core of the granule. Shear stress generated by aeration is also essential for mature granule formation.18 Oh et al. have suggested that there are three major steps in granule formation. First, according to the authors, physical movement is responsible for an initial self-aggregation among microbial cells. As a second step, microbial aggregations are stabilized by attractive forces such as Van der Waals forces. Finally, mature granules are formed by the help of extracellular polymeric substances (EPS) synthesis, growth of immobilized cells and hydrodynamic forces among others.19 Gao et al. (2011) have suggested that cell surface hydrophobicity is responsible for an initial cell-to-cell aggregation involved in granule formation.20 Recently, Zhang et al. (2017) have concluded that an alternating organic loading rate and starvation are crucial for stable granule formation.21 However, how do the alternating organic loading rate and starvation influence granule formation is not known. Further investigations on the role of these two parameters in EPS synthesis may provide valuable information on their role in granule formation. Thus, granule formation mechanism is still a subject of fundamental investigation.

Reactor and process parameters

Aerobic granules are usually developed during wastewater treatment using sequencing batch reactors (SBR).22,23 SBRs have well-defined operation cycle where each cycle could be of certain duration such as 3–6 hours. Each cycle is further divided into a definite filling period, aeration or mixing period, settling period and effluent drawing period.24 The operation cycle and its periods are the characteristics of SBR. Therefore, they are directly related to the mechanism behind granule formation. For instance, the short settling period of SBR is of great importance for stable granule formation. Qin et al. (2004) investigated the effect of the settling time on granule formation. Settling times of 5, 10, 15 and 20 minutes were evaluated. It was observed that a settling time of 5 minutes was most effective for granule development.25

The reactor height-to-diameter (H/D) ratio is another crucial parameter for aerobic granule formation. Awang et al. (2016) have demonstrated that an increase in the H/D ratio proportionally increases the maximum specific growth rate of the microorganisms of SBR.26 Linlin et al. (2005) have used a cylindrical reactor with a height-to-diameter (internal) ratio of 50 cm/17.6 cm.27 Similarly, Val del Río et al. (2012) have used a SBR for aerobic granule development which had a height-to-internal diameter ratio of 465 mm/85 mm.28 Usually the SBR used for aerobic granule formation are operated at a temperature of 15 to 27 °C.27–29 The activated sludge collected from the wastewater treatment plant is usually used as an inoculum for granule development.29,30 An influent COD of around 600 mg L−1 is effective for granule formation.29 The liquid volume exchange ratio of 50% is commonly used for each cycle of SBR used for aerobic granule formation.31 It is widely believed that periodic starvation imposed by the cyclic operation of SBR is a driving force of granule formation.32 The shear force generated by the up-flow air velocity is another process parameter to determine the quality of the granules.33 Tay et al. (2001) have found that when the supercritical up-flow air velocity was 0.3 cm s−1, small microbial flocs were dominants. However, by increasing this velocity to 1.2 cm s−1, dense aerobic granules were obtained.33 Likewise, Beun et al. (1999) have concluded that high shear force and short hydraulic retention time (HRT) are appropriate for aerobic granule formation.34

Thus, the process parameters such as settling time, HRT, up-flow air velocity, organic loading rate, starvation period and H/D ratio are some of the parameters directly related to granule formation. Most of these parameters have been optimized for compact and stable aerobic granule formation. However, the molecular biological mechanisms behind the effect of these parameters on granule formation are largely unexplored. There is a need to study the effect of these parameters on the microorganisms involved in granule formation. How the microorganism recognizes and responds to the changes in process parameters is a poorly understood subject. Further investigation of this topic would be useful for this technology.

Nutrient removal by aerobic granules

Nutrient (carbon, nitrogen and phosphorus) removal efficiency of aerobic granules is comparable to that of the conventional biological nutrient removal process. It has been claimed that the aerobic granules are capable of simultaneous nitrification, denitrification and phosphorus removal in a single step process involving only one reactor.35 This is a technical advantage of the aerobic granulation technology. During simultaneous nitrogen and phosphorus removal, nitrification and phosphorus removal could be seen even without proper granule development; however for efficient denitrification, the development of a mature granule is necessary.35 Mature granules have an anoxic zone at the center and most probably this anoxic zone is responsible for denitrification.35 Bao et al. (2009) have evaluated the effect of low temperature (10 °C) on aerobic granule development and nutrient removal.36 It has been reported that the granules with a diameter of around 3.4 mm could be formed in that temperature and the granules developed effective denitrification capability after maturation.36 A decrease in influent COD concentration was found to reduce the denitrification ability.36 These observations suggest that heterotrophic denitrification in the anoxic core of the granule was the main mechanism of denitrification. Apart from nitrification and subsequent heterotrophic denitrification, anaerobic ammonium oxidation (anammox) is a new possibility offered by aerobic granules. The anoxic/anaerobic core region of aerobic granules should support the growth of anammox bacteria. Theoretically, therefore, aerobic granules composed of the anammox bacteria should be suitable for the treatment of low strength wastewater such as municipal wastewater.

Kagawa et al. (2015) have designed a model to explain the nutrient removal performance of aerobic granules. The authors have concluded that the nutrient removal performance of aerobic granules depends on the DO concentration of the medium.37 Recently, Lashkarizadeh et al. (2016) have evaluated the effect of pH shock on the nutrient removal efficiency of aerobic granules. It was found that a high alkaline pH (pH 9) can irreversibly reduce the nitrogen removal efficiency from 88% to 66% and phosphorus removal efficiency from 98% and 50%.38 However, a mild acidic pH shock (pH 6) did not have any irreversible effect on the nutrient removal efficiency of the aerobic granules. Nutrient removal from low strength wastewater, such as municipal wastewater is a challenge for aerobic granules mostly because of the time taken for granule development. Kang et al. (2017) have evaluated the possibility of growing the granules in high strength wastewater and their subsequent application for nutrient removal from low strength wastewater. It has been concluded that maintaining an appropriate food-to-microorganism ratio would be crucial for the success of this strategy.39

Coma et al. (2012) have investigated the removal of phosphorus from domestic wastewater using aerobic granules. It was found that phosphorus removal by the granules could be inhibited by nitrite concentration higher than 5 mg nitrite-nitrogen L−1.40 Phosphorus is usually removed from wastewater by bioaccumulation using phosphorus-accumulating microorganisms.41 Apart from this common mechanism of phosphorus removal, aerobic granules are capable of removing phosphorus by precipitation. EDX-assisted SEM analysis has shown that phosphorus was precipitated in the core zone of aerobic granules mostly as Ca5(PO4)3OH. The report concluded that precipitation was responsible for removing as high as 45% of the total phosphorus removed by aerobic granules.42 Further studies would be needed to understand the phosphorus precipitation mechanism of aerobic granules. Factors involved in phosphorus precipitation and solubilisation dynamics need to be identified for a better understanding of this subject.

Biodegradation of pollutants

Since its discovery in the late 1990s, aerobic granulation technology has been successfully used for the biodegradation of a range of organic pollutants. Phenol is one of the organic pollutants efficiently degraded by aerobic granules. Adav et al. (2007) have demonstrated that as high as 1000 mg L−1 of phenol could be degraded by aerobic granules at an impressive degradation rate of 49 mg gVSS−1 h−1.43Candida tropicalis, a strain capable of degrading phenol at a high initial concentration, was isolated from the phenol-degrading aerobic granules. This strain was mostly detected in the surface layer of the granules.43 Yi et al. (2006) have developed p-nitrophenol-degrading aerobic granules. The granules were capable of degrading 40.1 mg L−1 of p-nitrophenol at a degradation rate of 19.3 mg gVSS−1 h−1.44 Aerobic granules grown in 500 mg L−1 of phenol have been shown to degrade as high as 250 to 2500 mg L−1 of pyridine. The maximum pyridine degradation rate was found to be around 73 mg gVSS−1 h−1.45 Basheer et al. (2012) have evaluated p-cresol biodegradation by aerobic granules. It has been reported that as high as 88% of 800 mg L−1 of p-cresol could be degraded by aerobic granules. A maximum p-cresol degradation rate of 960 mg gVSS−1 d−1 could be achieved when the initial p-cresol concentration was around 400 mg L−1.46

Similarly, the application of aerobic granules for the biodegradation of 4-chloroaniline has been reported by Zhu et al. (2011). A maximum specific degradation rate of 270 mg gVSS−1 d−1 could be achieved when the 4-chloroaniline concentration was more than 400 mg L−1. It has been reported that the granules were capable of completely removing around 8 g L−1 of 4-chloroaniline.47 Likewise, the application of aerobic granules for the biodegradation of Acid Red 18 has been evaluated by Sadri et al. (2016). A sequencing batch reactor with both anaerobic and aerobic phases was used for granule development and Acid Red 18 removal. It has been reported that around 50 mg L−1 of Acid Red 18 could be removed by the aerobic granules without any difficulty. However, an increase in the dye concentration from 50 to 100 mg L−1 was found to have a negative effect on the stability of the granules and their dye removal efficiency.48

From the above discussion, it can be concluded that aerobic granules are capable of degrading a range of pollutants. Various reports on pollutant biodegradation by aerobic granules suggest that in addition to municipal wastewater treatment, aerobic granulation technology would be effective for industrial wastewater treatment. Pollutant specific optimization of the process parameters might be needed for efficient removal. Concomitant biodegradation and adsorption seems to be responsible for the removal of pollutants such as dyes and relatively hydrophobic compounds.

Heavy metal removal by aerobic granules

Bio-sorption by aerobic granules is a potential tool to remove heavy metals from both municipal and industrial wastewater. Based on SEM-EDX (scanning electron microscope- energy dispersive X-ray) analysis, it has been reported that not only the surface, but the whole granule including the core zone can adsorb heavy metals.49 Pores found in the granules play a crucial role in the transfer of these pollutants into the inner zones of the granules. FTIR and XPS analyses showed that functional groups such as carboxylate, alcoholic and ether groups found in the aerobic granules would act as binding sites for heavy metals.49 It was found that the ratio between the initial heavy metal concentration and the initial aerobic granule concentration is one of the factors to determine the bio-sorption efficiency.50 Studies on Zn(II) bio-sorption showed that a maximum of 270 mg of Zn(II) could be adsorbed by each g of aerobic granules. Thus, it could be an effective tool for industrial wastewater treatment.50 Luo et al. (2016) investigated Cu(II) bio-sorption by aerobic granules. It has been concluded that complex formation and ion exchange were the two major mechanisms of Cu(II) bio-sorption. The distribution of Cu(II) on the granule surface was following a similar trend to that of the metal ions usually found in the granules such as Ca2+ and Mg2+. pH and ionic strength were found to have a beneficial effect on Cu(II) bio-sorption by aerobic granules.51

Both bacteria and fungi are involved in aerobic granule formation. Based on process parameters, either bacteria or the fungi could be the dominant microbial community of the granule. Wang et al. (2015) compared the heavy metal removal potential of bacteria-dominated and fungi-dominated granules.52 It was reported that both these granules could be used as bio-sorbent for Zn(II), Cu(II) and Ni(II) removal. However, they were not effective in Sb(V). It was observed that the surface modification of both the granule types using Fe(III) could improve their Sb(V) removal efficiency.52 Nancharaiah et al. (2006) have demonstrated that aerobic granules are capable of uranium bio-sorption. It was observed that the bio-sorption of uranium was followed by a release of Ca2+, Mg2+, Na+ and K+ to the liquid phase. It indicates that there might be an ion exchange mechanism involved in uranium bio-sorption by aerobic granules.53 From the above discussion, it is evident that aerobic granules could be an effective bio-sorption tool for heavy metal removal from municipal as well as industrial wastewater.

Full-scale real wastewater treatment using aerobic granules

Domestic wastewater treatment in an aerobic granule-based full-scale wastewater plant has been demonstrated by Pronk et al. (2015). The study was conducted in Garmerwolde wastewater treatment plant of the Netherlands.54 It took around 5 months to develop a sludge bed with mature granules and the biomass concentration of the bed was as high as 8 g L−1. The granular sludge bed was effective in nitrogen and phosphorus removal both in summer and winter seasons.54 It was capable of maintaining the effluent nitrogen and phosphorus levels below the recommended limit of 7 mg L−1 and 1 mg L−1, respectively. Moreover, the energy requirement of the full-scale aerobic granule-based process was around 58% to 63% less than that of the common activated sludge-based process.54 Li et al. (2014) have demonstrated wastewater treatment by aerobic granules in a full-scale SBR with a working volume of 50000 m3 d−1.55 Mature aerobic granules were obtained after around 337 days of operation of the full-scale reactor. Compared to traditional wastewater treatment processes, the aerobic granular sludge developed in the full-scale reactor was denser with a better settling ability. The average diameter of these granules was around 0.5 mm.55

Stubbé et al. (2016) have investigated phosphorus precipitation within the aerobic granules developed in a full-scale aerobic granular sludge process.56 XRD (X-ray diffraction) analysis confirmed the presence of SiO4 (quartz) crystals inside the granules. However, the mature granules did not have any significant amount of precipitated phosphorus. It has been concluded that phosphorus precipitation and dissolution is a dynamic process, and at a different point of a cycle of SBR operation, the amount of precipitated phosphorus found in the granules could be different.56 According to Niermans et al. (2014), 20 full-scale aerobic granular sludge processes, under the brand name Nereda®, are in different stages of construction in different countries including the UK, Australia, Switzerland, Netherlands and Brazil among others.57 A full-scale Nereda process can offer an excellent effluent quality with less than 5 mg L−1 of total nitrogen and around 0.3 mg L−1 of total phosphorus. Additionally, the Nereda process can save energy by around 40%.57 Thus, a few full-scale aerobic granule-based processes have been successfully installed for real wastewater treatment. Further information on the design, cost, process conditions and efficiency of these treatment plants will be useful for the rapid propagation of this technology.

Challenges of aerobic granulation technology

It is not possible to develop a process where 100% of the sludge is in granular form. For instance, a process may have around 50% of the sludge as proper granules and the remainder may be found as dense microbial flocs. A process with 100% granular sludge is expected to have better settling ability with very low sludge volume index (SVI). Increasing the total percentage of proper granules in the granular sludge is still a challenge for this technology. SBR is the most suitable reactor for aerobic granule development. Almost all studies on aerobic granules reported so far were conducted using SBR. However, for real wastewater treatment, continuous-flow reactors are mostly used all over the world.58 Therefore, development of aerobic granules in a full-scale continuous-flow reactor can be considered for further investigation. Granules are microspheres of mixed microorganisms. It takes a few weeks to months to develop the granules. However, sometimes granules are not stable enough to withstand the changes in process conditions.59–61 Thus, disintegration of the granules due to unknown process conditions is a challenge which needs further investigation.62–66

Aerobic granulation technology is a new wastewater treatment technology. It has been predicted that in the future, it would replace the conventional activated sludge process used for wastewater treatment. However, the reactors presently being used for the activated sludge process are not suitable for aerobic granulation technology. Thus, converting an existing activated sludge process to an aerobic granulation process would be a challenge.67 This aspect needs further investigation. At a relatively low dissolved oxygen concentration, aerobic granules can remove nitrogen through simultaneous nitrification and denitrification. However, a low oxygen concentration is not suitable for granule formation. Thus, it is a challenge to grow the granules at a low dissolved oxygen concentration.68 The aerobic granule development process takes several weeks to a few months. During the granule development period, the nutrient removal efficiency is usually poor.69 Therefore, a long granulation time is another challenge for the real application of the aerobic granulation technology.70 Further optimization of the engineering strategies and fundamental molecular microbiological investigations would be crucial for dealing with the challenges of this technology.

Conclusions

Aerobic granulation is a new technology which has many advantages over activated sludge-based conventional wastewater treatment technology. The commercial application of this technology has been started and a few full-scale aerobic granule-based wastewater treatment plants have been installed. Aerobic granule formation mechanism is still a mystery. There are different hypotheses to explain the mechanism; however, there is no strong experimental evidence to establish them. Both fungi and bacteria are involved in aerobic granule formation and usually the bacteria are the dominant microorganisms. Sequencing batch reactors are commonly used for this technology. However, continuous-flow reactors would be more appropriate for its widespread application. Aerobic granulation technology is more appropriate for the treatment of high-strength industrial wastewater. For the treatment of low-strength domestic wastewater, it will be necessary to increase its COD by the addition of external carbon sources such as volatile fatty acids. Aerobic granules have excellent nutrient removal efficiency. Aerobic granules can remove phosphorus by precipitation within the granules. The exact mechanism of phosphorus precipitation by the aerobic granules is still a subject of fundamental research. Apart from nutrient removal, aerobic granules are efficient in the biodegradation of organic pollutants such as phenol and heavy metal removal. Granule formation is a time-consuming process and the unpredictable disintegration of the granules is a challenge for this technology. Fundamental microbiological investigations would be crucial to deal with this issue. Quorum sensing and quorum quenching are two important molecular microbiological mechanisms which may have a direct influence on granule formation and stability. Therefore, further investigation of these subjects would give valuable information on granule formation mechanisms.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

The authors are thankful to the City of Calgary and the Natural Sciences and Engineering Research Council-Industrial Research Chair (NSERC-IRC) of Canada for financial assistance.